Extracts from Dendropanax morbifera leaves ameliorates cerebral ischemia-induced hippocampal damage by reducing oxidative damage in gerbil.

AIM: In this study, we investigated the effects of Dendropanax morbifera extract (DME) on neuroprotection against ischemic damage in gerbils. METHODS: DME (100 or 300 mg/kg) was orally administered to gerbils for three weeks, and 2 h after the last DME treatment, transient forebrain ischemia in the common carotid arteries was induced for 5 min. The forebrain ischemia-related cognitive impairments were assessed by spontaneous motor activity and passive avoidance test one and four days after ischemia, respectively. In addition, surviving and degenerating neurons were morphologically confirmed by neuronal nuclei immunohistochemical staining and Fluoro-Jade C staining, respectively, four days after ischemia. Changes of glial morphology were visualized by immunohistochemical staining for each marker such as glial fibrillary acidic protein and ionized calcium-binding protein. Oxidative stress was determined by measurements of dihydroethidium, O2· (formation of formazan) and malondialdehyde two days after ischemia. In addition, glutathione redox system such as reduced glutathione, oxidized glutathione levels, glutathione peroxidase, and glutathione reductase activities were measured two days after ischemia. RESULTS: Spontaneous motor activity monitoring and passive avoidance tests showed that treatment with 300 mg/kg DME, but not 100 mg/kg, significantly alleviated ischemia-induced memory impairments. In addition, approximately 67 % of mature neurons survived and 29.3 % neurons were degenerated in hippocampal CA1 region four days after ischemia, and ischemia-induced morphological changes in astrocytes and microglia were decreased in the CA1 region after 300 mg/kg DME treatment. Furthermore, treatment with 300 mg/kg DME significantly ameliorated ischemia-induced oxidative stress, such as superoxide formation and lipid peroxidation, two days after ischemia. In addition, ischemia-induced reduction of the glutathione redox system in the hippocampus, assessed two days after the ischemia, was ameliorated by treatment with 300 mg/kg DME. These suggest that DME can potentially reduce ischemia-induced neuronal damage through its antioxidant properties.

Cu,Zn-Superoxide Dismutase has Minimal Effects Against Cuprizone-Induced Demyelination, Microglial Activation, and Neurogenesis Defects in the C57BL/6 Mouse Hippocampus.

Cuprizone causes consistent demyelination and oligodendrocyte damage in the mouse brain. Cu,Zn-superoxide dismutase 1 (SOD1) has neuroprotective potential against various neurological disorders, such as transient cerebral ischemia and traumatic brain injury. In this study, we investigated whether SOD1 has neuroprotective effects against cuprizone-induced demyelination and adult hippocampal neurogenesis in C57BL/6 mice, using the PEP-1-SOD1 fusion protein to facilitate the delivery of SOD1 protein into hippocampal neurons. Eight weeks feeding of cuprizone-supplemented (0.2%) diets caused a significant decrease in myelin basic protein (MBP) expression in the stratum lacunosum-moleculare of the CA1 region, the polymorphic layer of the dentate gyrus, and the corpus callosum, while ionized calcium-binding adapter molecule 1 (Iba-1)-immunoreactive microglia showed activated and phagocytic phenotypes. In addition, cuprizone treatment reduced proliferating cells and neuroblasts as shown using Ki67 and doublecortin immunostaining. Treatment with PEP-1-SOD1 to normal mice did not show any significant changes in MBP expression and Iba-1-immunoreactive microglia. However, Ki67-positive proliferating cells and doublecortin-immunoreactive neuroblasts were significantly decreased. Simultaneous treatment with PEP-1-SOD1 and cuprizone-supplemented diets did not ameliorate the MBP reduction in these regions, but mitigated the increase of Iba-1 immunoreactivity in the corpus callosum and alleviated the reduction of MBP in corpus callosum and proliferating cells, not neuroblasts, in the dentate gyrus. In conclusion, PEP-1-SOD1 treatment only has partial effects to reduce cuprizone-induced demyelination and microglial activation in the hippocampus and corpus callosum and has minimal effects on proliferating cells in the dentate gyrus.

Tat-CCT2 Protects the Neurons from Ischemic Damage by Reducing Oxidative Stress and Activating Autophagic Removal of Damaged Protein in the Gerbil Hippocampus.

CCT2 is a eukaryotic chaperonin TCP-1 ring complex subunit that mediates protein folding, autophagosome incorporation, and protein aggregation. In this study, we investigated the effects of CCT on oxidative and ischemic damage using in vitro and in vivo experimental models. The Tat-CCT2 fusion protein was efficiently delivered into HT22 cells in a concentration- and time-dependent manner, and the delivered protein was gradually degraded in HT22 cells. Incubation with Tat-CCT2 significantly ameliorated the 200 µM hydrogen peroxide (H2O2)-induced reduction in cell viability in a concentration-dependent manner, and 8 µM Tat-CCT2 treatment significantly alleviated H2O2-induced DNA fragmentation and reactive oxygen species formation in HT22 cells. In gerbils, CCT2 protein was efficiently delivered into pyramidal cells in CA1 region by intraperitoneally injecting 0.5 mg/kg Tat-CCT2, as opposed to control CCT2. In addition, treatment with 0.2 or 0.5 mg/kg Tat-CCT2 mitigated ischemia-induced hyperlocomotive activity 1 d after ischemia and confirmed the neuroprotective effects by NeuN immunohistochemistry in the hippocampal CA1 region 4 d after ischemia. Tat-CCT2 treatment significantly reduced the ischemia-induced activation of astrocytes and microglia in the hippocampal CA1 region 4 d after ischemia. Furthermore, treatment with 0.2 or 0.5 mg/kg Tat-CCT2 facilitated ischemia-induced autophagic activity and ameliorated ischemia-induced autophagic initiation in the hippocampus 1 d after ischemia based on western blotting for LC3B and Beclin-1, respectively. Levels of p62, an autophagic substrate, significantly increased in the hippocampus following treatment with Tat-CCT2. These results suggested that Tat-CCT2 exerts neuroprotective effects against oxidative stress and ischemic damage by promoting the autophagic removal of damaged proteins or organelles.

Comparison of the Effects of Cuprizone on Demyelination in the Corpus Callosum and Hippocampal Progenitors in Young Adult and Aged Mice.

Cuprizone is commonly used to induce neuronal demyelination in mice. In the present study, we compared the cuprizone-induced demyelination in the corpus callosum and investigated the effects of cuprizone on proliferating cells and neuroblasts in the dentate gyrus of young adult and aged mice. 5-week- and 23-month-old mice were fed a normal diet or a 0.2% cuprizone-enriched diet for 5 weeks. Mice fed a cuprizone-supplemented diet showed a significant reduction in myelin basic protein-positive structures in the corpus callosum, with the reduction in myelinated fibers being confirmed by electron microscopic analysis. In addition, we observed a marked increase in Ki67-positive proliferating cells and doublecortin-immunoreactive neuroblasts in young adult mice in response to cuprizone treatment, although not in aged mice, as the basal levels of these cells were significantly lower in these older mice. Furthermore, Ser133-phosphorylated cAMP response element-binding protein (pCREB)-positive nuclei and brain-derived neurotrophic factor (BDNF) protein levels were significantly reduced in young adult mice following cuprizone treatment in young adult, although again not in the aged mice. However, in both young adult and aged mice, there were no significant reductions in hippocampal mature neurons in response to cuprizone treatment. These observations indicate that in the mice of both age groups a cuprizone-supplemented diet contributes to an increase in demyelination in the corpus callosum and neural progenitor cells in the dentate gyrus, although the damage is more pronounced in young adult mice. This demyelination and reduction in neural progenitor cells may be associated with changes in the levels of BDNF and pCREB in the dentate gyrus.

Tat-p27 Ameliorates Neuronal Damage Reducing α-Synuclein and Inflammatory Responses in Motor Neurons After Spinal Cord Ischemia.

p27Kip1 (p27) regulates the cell cycle by inhibiting G1 progression in cells. Several studies have shown conflicting results on the effects of p27 against cell death in various insults. In the present study, we examined the neuroprotective effects of p27 against H2O2-induced oxidative stress in NSC34 cells and against spinal cord ischemia-induced neuronal damage in rabbits. To promote delivery into NSC34 cells and motor neurons in the spinal cord, Tat-p27 fusion protein and its control protein (Control-p27) were synthesized with or without Tat peptide, respectively. Tat-p27, but not Control-27, was efficiently introduced into NSC34 cells in a concentration- and time-dependent manner, and the protein was detected in the cytoplasm. Tat-p27 showed neuroprotective effects against oxidative stress induced by H2O2 treatment and reduced the formation of reactive oxygen species, DNA fragmentation, and lipid peroxidation in NSC34 cells. Tat-p27, but not Control-p27, ameliorated ischemia-induced neurological deficits and cell damage in the rabbit spinal cord. In addition, Tat-p27 treatment reduced the expression of α-synuclein, activation of microglia, and release of pro-inflammatory cytokines such as interleukin-1ß and tumor necrosis factor-α in the spinal cord. Taken together, these results suggest that Tat-p27 inhibits neuronal damage by decreasing oxidative stress, α-synuclein expression, and inflammatory responses after ischemia.

Neuropathological changes in dorsal root ganglia induced by pyridoxine in dogs.

BACKGROUND: Pyridoxine (PDX; vitamin B6), is an essential vitamin. PDX deficiency induces various symptoms, and when PDX is misused it acts as a neurotoxicant, inducing severe sensory neuropathy. RESULTS: To assess the possibility of creating a reversible sensory neuropathy model using dogs, 150 mg/kg of PDX was injected subcutaneously into dogs for 7 days and body weight measurements, postural reaction assessments, and electrophysiological recordings were obtained. In addition, the morphology of dorsal root ganglia (DRG) and changes in glial fibrillary acidic protein (GFAP) immunoreactive satellite glial cells and ionized calcium-binding adapter molecule 1 (Iba-1) immunoreactive microglia/macrophages were assessed at 1 day, 1 week, and 4 weeks after the last PDX treatment. During the administration period, body weight and proprioceptive losses occurred. One day after the last PDX treatment, electrophysiological recordings showed the absence of the H-reflex in the treated dogs. These phenomena persisted over the four post-treatment weeks, with the exception of body weight which recovered to the pre-treatment level. Staining (CV and HE) results revealed significant losses of large-sized neurons in the DRG at 1 day and 1 week after PDX treatment cessation, but the losses were recovered at 4 weeks post-treatment. The Iba-1 and GFAP immunohistochemistry results showed pronounced increases in reactive microglia/macrophage and satellite glial cell at 1 day and 1 week, respectively, after the last PDX treatment, and thereafter, immunoreactivity decreased with increasing time after PDX treatment. CONCLUSIONS: The results suggest that PDX-induced neuropathy is reversible in dogs; thus, dogs can be considered a good experimental model for research on neuropathy.

Ischemia-related changes of fat-mass and obesity-associated protein expression in the gerbil hippocampus.

Fat-mass and obesity-associated protein (Fto) plays important roles in energy metabolism. It also acts as a demethylase and is most abundantly found in the brain. In the present study, we examined the spatial and temporal changes of Fto immunoreactivity after five minutes of transient forebrain ischemia in the hippocampus. In the control group, Fto immunoreactivity was mainly observed in the nucleus of pyramidal cells in the CA1 and CA3 regions as well as the polymorphic layer, granule cell layer, and subgranular zone of the dentate gyrus. Fto immunoreactivity was transiently, but not significantly, increased in the hippocampal CA3 region and the dentate gyrus two days after ischemia compared to mice without ischemia in the sham-operated group. Four days after ischemia, low Fto immunoreactivity was observed in the stratum pyramidale of the CA1 region because of neuronal death, but Fto immunoreactive cells were abundantly detected in the stratum pyramidale of the CA3 region, which is relatively resistant to ischemic damage. Thereafter, Fto immunoreactivity progressively decreased in the hippocampal CA1 and CA3 regions and the dentate gyrus until ten days after ischemia. At this time-point, Fto immunoreactivity was significantly lower in the hippocampal CA1 and CA3 regions and the dentate gyrus compared to that in the sham-operated group. The reduction of Fto immunoreactive structures in the hippocampus may be associated with impairments in Fto-related hippocampal function.

Physical Stress Induced Reduction of Proliferating Cells and Differentiated Neuroblasts Is Ameliorated by Fermented Laminaria japonica Extract Treatment.

Laminaria japonica is widely cultivated in East Asia, including South Korea. Fucoidan, a main component of L. japonica, protects neurons from neurological disorders such as ischemia and traumatic brain injury. In the present study, we examined the effects of extract from fermented L. japonica on the reduction of proliferating cells and neuroblasts in mice that were physically (with electric food shock) or psychologically (with visual, auditory and olfactory sensation) stressed with the help of a communication box. Vehicle (distilled water) or fermented L. japonica extract (50 mg/kg) were orally administered to the mice once a day for 21 days. On the 19th day of the treatment, physical and psychological stress was induced by foot shock using a communication box and thereafter for three days. Plasma corticosterone levels were significantly increased after exposure to physical stress and decreased Ki67 positive proliferating cells and doublecortin immunoreactive neuroblasts. In addition, western blot analysis demonstrated that physical stress as well as psychological stress decreased the expression levels of brain-derived neurotrophic factor (BDNF) and the number of phosphorylated cAMP response element binding protein (pCREB) positive nuclei in the dentate gyrus. Fermentation of L. japonica extract significantly increased the contents of reduced sugar and phenolic compounds. Supplementation with fermented L. japonica extract significantly ameliorated the increases of plasma corticosterone revels and decline in the proliferating cells, neuroblasts, and expression of BDNF and pCREB in the physically stressed mice. These results indicate that fermented L. japonica extract has positive effects in ameliorating the physical stress induced reduction in neurogenesis by modulating BDNF and pCREB expression in the dentate gyrus.

P27 Protects Neurons from Ischemic Damage by Suppressing Oxidative Stress and Increasing Autophagy in the Hippocampus.

p27Kip1 (p27), a well-known cell regulator, is involved in the regulation of cell death and survival. In the present study, we observed the effects of p27 against oxidative stress induced by H2O2 in HT22 cells and transient ischemia in gerbils. Tat (trans-acting activator of transcription) peptide and p27 fusion proteins were prepared to facilitate delivery into cells and across the blood-brain barrier. The tat-p27 fusion protein, rather than its control protein Control-p27, was delivered intracellularly in a concentration and incubation time-dependent manner and showed its activity in HT22 cells. The localization of the delivered Tat-p27 protein was also confirmted in the HT22 cells and hippocampus in gerbils. In addition, the optimal concentration (5 µM) of Tat-p27 was determined to protect neurons from cell death induced by 1 mM H2O2. Treatment with 5 µM Tat-p27 significantly ameliorated H2O2-induced DNA fragmentation and the formation of reactive oxygen species (ROS) in HT22 cells. Tat-p27 significantly mitigated the increase in locomotor activity a day after ischemia and neuronal damage in the hippocampal CA1 region. It also reduced the ischemia-induced membrane phospholipids and ROS formation. In addition, Tat-p27 significantly increased microtubule-associated protein 1A/1B light chain 3A/3B expression and ameliorated the H2O2 or ischemia-induced increases of p62 and decreases of beclin-1 in the HT22 cells and hippocampus. These results suggest that Tat-p27 protects neurons from oxidative or ischemic damage by reducing ROS-induced damage and by facilitating the formation of autophagosomes in hippocampal cells.

Phosphoglycerate Mutase 1 Prevents Neuronal Death from Ischemic Damage by Reducing Neuroinflammation in the Rabbit Spinal Cord.

Phosphoglycerate mutase 1 (PGAM1) is a glycolytic enzyme that increases glycolytic flux in the brain. In the present study, we examined the effects of PGAM1 in conditions of oxidative stress and ischemic damage in motor neuron-like (NSC34) cells and the rabbit spinal cord. A Tat-PGAM1 fusion protein was prepared to allow easy crossing of the blood-brain barrier, and Control-PGAM1 was synthesized without the Tat peptide protein transduction domain. Intracellular delivery of Tat-PGAM1, not Control-PGAM1, was achieved in a time- and concentration-dependent manner. Immunofluorescent staining confirmed the intracellular expression of Tat-PGAM1 in NSC34 cells. Tat-PGAM1, but not Control-PGAM1, significantly alleviated H2O2-induced oxidative stress, neuronal death, mitogen-activated protein kinase, and apoptosis-inducing factor expression in NSC34 cells. After ischemia induction in the spinal cord, Tat-PGAM1 treatment significantly improved ischemia-induced neurological impairments and ameliorated neuronal cell death in the ventral horn of the spinal cord 72 h after ischemia. Tat-PGAM1 treatment significantly mitigated the ischemia-induced increase in malondialdehyde and 8-iso-prostaglandin F2α production in the spinal cord. In addition, Tat-PGAM1, but not Control-PGAM1, significantly decreased microglial activation and secretion of pro-inflammatory cytokines, such as interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α induced by ischemia in the ventral horn of the spinal cord. These results suggest that Tat-PGAM1 can be used as a therapeutic agent to reduce spinal cord ischemia-induced neuronal damage by lowering the oxidative stress, microglial activation, and secretion of pro-inflammatory cytokines, such as IL-1ß, IL-6, and TNF-α.

Pyridoxine Deficiency Exacerbates Neuronal Damage after Ischemia by Increasing Oxidative Stress and Reduces Proliferating Cells and Neuroblasts in the Gerbil Hippocampus.

We investigated the effects of pyridoxine deficiency on ischemic neuronal death in the hippocampus of gerbil (n = 5 per group). Serum pyridoxal 5'-phosphate levels were significantly decreased in Pyridoxine-deficient diet (PDD)-fed gerbils, while homocysteine levels were significantly increased in sham- and ischemia-operated gerbils. PDD-fed gerbil showed a reduction in neuronal nuclei (NeuN)-immunoreactive neurons in the medial part of the hippocampal CA1 region three days after. Reactive astrocytosis and microgliosis were found in PDD-fed gerbils, and transient ischemia caused the aggregation of activated microglia in the stratum pyramidale three days after ischemia. Lipid peroxidation was prominently increased in the hippocampus and was significantly higher in PDD-fed gerbils than in Control diet (CD)-fed gerbils after ischemia. In contrast, pyridoxine deficiency decreased the proliferating cells and neuroblasts in the dentate gyrus in sham- and ischemia-operated gerbils. Nuclear factor erythroid-2-related factor 2 (Nrf2) and brain-derived neurotrophic factor (BDNF) levels also significantly decreased in PDD-fed gerbils sham 24 h after ischemia. These results suggest that pyridoxine deficiency accelerates neuronal death by increasing serum homocysteine levels and lipid peroxidation, and by decreasing Nrf2 levels in the hippocampus. Additionally, it reduces the regenerated potentials in hippocampus by decreasing BDNF levels. Collectively, pyridoxine is an essential element in modulating cell death and hippocampal neurogenesis after ischemia.

Phosphoglycerate Mutase 1 Promotes Cell Proliferation and Neuroblast Differentiation in the Dentate Gyrus by Facilitating the Phosphorylation of cAMP Response Element-Binding Protein.

In a previous study, we observed a significant increase in phosphoglycerate mutase 1 (PGAM1) levels after pyridoxine treatment. In the present study, we investigated the effects of PGAM1 on novel object recognition, cell proliferation, and neuroblast differentiation in the dentate gyrus. We generated a Tat-PGAM1 fusion protein to cross the blood-brain barrier and neuronal plasma membrane. We administered the Tat peptide, control-PGAM1, or Tat-PGAM1 fusion protein to 8-week-old mice once a day for 3 weeks and tested novel object recognition memory. The mice were then euthanized to conduct western blot analysis for polyhistidine expression and immunohistochemical analysis for Ki67, doublecortin, and phosphorylated cAMP response element-binding protein. Mice treated with Tat peptide showed similar exploration times for familiar and new objects and the discrimination index was significantly lower in this group than in the control group. Tat-PGAM1 moderately increased the exploration time of new objects when compared to familiar objects, while the discrimination index was significantly higher in the Tat-PGAM1-treated group, but not in the control-PGAM1-treated group, when compared with the control group. Higher PGAM1 protein expression was observed in the hippocampus of Tat-PGAM1-treated mice when compared with the hippocampi of control, Tat peptide-, and control-PGAM1-treated mice, using western blot analysis. In addition, the numbers of proliferating cells and differentiated neuroblasts were significantly lower in the Tat peptide-treated group than in the control group. In contrast, the numbers of proliferating cells and differentiated neuroblasts in the dentate gyrus were higher in the Tat-PGAM1-treated group than in the control group. Administration of Tat-PGAM1 significantly facilitated the phosphorylation of cAMP response element-binding protein in the dentate gyrus. Administration of control-PGAM1 did not show any significant effects on novel object recognition, cell proliferation, and neuroblast differentiation in the dentate gyrus. These results suggest that PGAM1 plays a role in cell proliferation and neuroblast differentiation in the dentate gyrus via the phosphorylation of cAMP response element-binding protein in the hippocampus.

Differential regional infarction, neuronal loss and gliosis in the gerbil cerebral hemisphere following 30 min of unilateral common carotid artery occlusion.

The degree of transient ischemic damage in the cerebral hemisphere is different according to duration of transient ischemia and cerebral regions. Mongolian gerbils show various lesions in the hemisphere after transient unilateral occlusion of the common carotid artery (UOCCA) because they have different types of patterns of anterior and posterior communicating arteries. We examined differential regional damage in the ipsilateral hemisphere of the gerbil after 30 min of UOCCA by using 2,3,5-triphenyltetrazolium chloride (TTC) staining, cresyl violet (CV) Nissl staining, Fluoro-Jade B (F-J B) fluorescence staining, and NeuN immunohistochemistry 5 days after UOCCA. In addition, regional differences in reactions of astrocytes and microglia were examined using GFAP and Iba-1 immunohistochemistry. After right UOCCA, neurological signs were assessed to define ischemic symptomatic animals. Moderate symptomatic gerbils showed several infarcts, while mild symptomatic gerbils showed selective neuronal death/loss in the primary motor and sensory cortex, striatum, thalamus, and hippocampus 5 days after UOCCA. In the areas, morphologically changed GFAP immunoreactive astrocytes and Iba-1 immunoreactive microglia were found, and their numbers were increased or decreased according to the damaged areas. In brief, our results demonstrate that 30 min of UOCCA in gerbils produced infarcts or selective neuronal death depending on ischemic severity in the ipsilateral cerebral cortex, striatum, thalamus and hippocampus, showing that astrocytes and microglia were differently reacted 5 days after UOCCA. Taken together, a gerbil model of 30 min of UOCCA can be used to study mechanisms of infarction and/or regional selective neuronal death/loss as well as neurological dysfunction following UOCCA.

Leaf extracts from Dendropanax morbifera Léveille mitigate mercury-induced reduction of spatial memory, as well as cell proliferation, and neuroblast differentiation in rat dentate gyrus.

BACKGROUND: The brain is susceptible to methylmercury toxicity, which causes irreversible damage to neurons and glia and the leaf extract Dendropanax morbifera Léveille (DML) has various biological functions in the nervous system. In this study, we examined the effects of DML on mercury-induced proliferating cells and differentiated neuroblasts. METHODS: Dimethylmercury (5 µg/kg) and galantamine (5 mg/kg) was administered intraperitoneally and/or DML (100 mg/kg) was orally to 7-week-old rats every day for 36 days. One hour after the treatment, novel object recognition test was examined. In addition, spatial probe tests were conducted on the 6th day after 5 days of continuous training in the Morris swim maze. Thereafter, the rats were euthanized for immunohistochemical staining analysis with Ki67 and doublecortin and measurement for acetylcholinesterase (AChE) activity. RESULTS: Dimethylmercury-treated rats showed reduced discrimination index in novel object recognition test and took longer to find the platform than did control group. Compared with dimethylmercury treatment alone, supplementation with DML or galatamine significantly ameliorated the reduction of discrimination index and reduced the time spent to find the platform. In addition, the number of platform crossings was lower in the dimethylmercury-treated group than in controls, while the administration of DML or galantamine significantly increased the number of crossings than did dimethylmercury treatment alone. Proliferating cells and differentiated neuroblasts, assessed by Ki67 and doublecortin immunohistochemical staining was significantly decreased in the dimethylmercury treated group versus controls. Supplementation with DML or galantamine significantly increased the number of proliferating cells and differentiated neuroblasts in the dentate gyrus. In addition, treatment with dimethylmercury significantly increased AChE activity in hippocampal homogenates, while treatment with dimethylmercury+DML or dimethylmercury+galantamine significantly ameliorated this increase. CONCLUSIONS: These results suggest that DML may be a functional food that improves dimethylmercury-induced memory impairment and ameliorates dimethylmercury-induced reduction in proliferating cells and differentiated neuroblasts, and demonstrates corresponding activation of AChE activity in the dentate gyrus.

Fate of Astrocytes in The Gerbil Hippocampus After Transient Global Cerebral Ischemia.

Neuronal death and reactive gliosis are major features of brain tissue damage following transient global cerebral ischemia (tgCI). This study investigated long-term changes in neuronal death and astrogliosis in the gerbil hippocampus for 180 days after 5 min of tgCI. A massive loss of pyramidal neurons was found in the hippocampal CA1 area (CA1) area between 5 and 30 days after tgCI by Fluoro-Jade B (FJB, a marker for neuronal degeneration) histofluorescence staining, but pyramidal neurons in the CA2/3 area did not die. The reaction of astrocytes (astrogliosis) was examined by glial fibrillary acidic protein (GFAP) immunohistochemistry. Morphological change or degeneration (death) of the astrocytes was found in the CA1 area after tgCI, but, in the CA2/3 area, astrogliosis was hardly shown. GFAP immunoreactive astrocytes in the CA1 area was significantly increased in number with time and peaked at 30 days after tgCI, and they began to be degenerated or dead from 40 days after tgCI. The effect was examined by double immunofluorescence staining for FJB and GFAP. The number of FJB/GFAP⁺ cells (degenerating astrocytes) was gradually increased with time after tgCI. At 180 days after tgCI, FJB/GFAP⁺ cells were significantly decreased, but FJB⁺ cells (dead astrocytes) were significantly increased. In brief, 5 min of tgCI induced a progressive degeneration of CA1 pyramidal neurons from 5 until 30 days with an increase of reactive astrocytes, and, thereafter, astrocytes were degenerated with time and dead at later times. This phenomenon might be shown due to the death of neurons.

Risperidone Treatment after Transient Ischemia Induces Hypothermia and Provides Neuroprotection in the Gerbil Hippocampus by Decreasing Oxidative Stress.

Compelling evidence from preclinical and clinical studies has shown that mild hypothermia is neuroprotective against ischemic stroke. We investigated the neuroprotective effect of post-risperidone (RIS) treatment against transient ischemic injury and its mechanisms in the gerbil brain. Transient ischemia (TI) was induced in the telencephalon by bilateral common carotid artery occlusion (BCCAO) for 5 min under normothermic condition (37 ± 0.2 °C). Treatment of RIS induced hypothermia until 12 h after TI in the TI-induced animals under uncontrolled body temperature (UBT) compared to that under controlled body temperature (CBT) (about 37 °C). Neuroprotective effect was statistically significant when we used 5 and 10 mg/kg doses (p < 0.05, respectively). In the RIS-treated TI group, many CA1 pyramidal neurons of the hippocampus survived under UBT compared to those under CBT. In this group under UBT, post-treatment with RIS to TI-induced animals markedly attenuated the activation of glial cells, an increase of oxidative stress markers [dihydroethidium, 8-hydroxy-2' -deoxyguanosine (8-OHdG), and 4-Hydroxynonenal (4-HNE)], and a decrease of superoxide dismutase 2 (SOD2) in their CA1 pyramidal neurons. Furthermore, RIS-induced hypothermia was significantly interrupted by NBOH-2C-CN hydrochloride (a selective 5-HT2A receptor agonist), but not bromocriptine mesylate (a D2 receptor agonist). Our findings indicate that RIS-induced hypothermia can effectively protect neuronal cell death from TI injury through attenuation of glial activation and maintenance of antioxidants, showing that 5-HT2A receptor is involved in RIS-induced hypothermia. Therefore, RIS could be introduced to reduce body temperature rapidly and might be applied to patients for hypothermic therapy following ischemic stroke.

Effects of Scopolamine and Melatonin Cotreatment on Cognition, Neuronal Damage, and Neurogenesis in the Mouse Dentate Gyrus.

It has been demonstrated that melatonin plays important roles in memory improvement and promotes neurogenesis in experimental animals. We examined effects of melatonin on cognitive deficits, neuronal damage, cell proliferation, neuroblast differentiation and neuronal maturation in the mouse dentate gyrus after cotreatment of scopolamine (anticholinergic agent) and melatonin. Scopolamine (1 mg/kg) and melatonin (10 mg/kg) were intraperitoneally injected for 2 and/or 4 weeks to 8-week-old mice. Scopolamine treatment induced significant cognitive deficits 2 and 4 weeks after scopolamine treatment, however, cotreatment of scopolamine and melatonin significantly improved spatial learning and short-term memory impairments. Two and 4 weeks after scopolamine treatment, neurons were not damaged/dead in the dentate gyrus, in addition, no neuronal damage/death was shown after cotreatment of scopolamine and melatonin. Ki67 (a marker for cell proliferation)- and doublecortin (a marker for neuroblast differentiation)-positive cells were significantly decreased in the dentate gyrus 2 and 4 weeks after scopolamine treatment, however, cotreatment of scopolamine and melatonin significantly increased Ki67- and doublecortin-positive cells compared with scopolamine-treated group. However, double immunofluorescence for NeuN/BrdU, which indicates newly-generated mature neurons, did not show double-labeled cells (adult neurogenesis) in the dentate gyrus 2 and 4 weeks after cotreatment of scopolamine and melatonin. Our results suggest that melatonin treatment recovers scopolamine-induced spatial learning and short-term memory impairments and restores or increases scopolamine-induced decrease of cell proliferation and neuroblast differentiation, but does not lead to adult neurogenesis (maturation of neurons) in the mouse dentate gyrus following scopolamine treatment.

Neuronal loss and gliosis in the rat striatum subjected to 15 and 30 minutes of middle cerebral artery occlusion.

Selective neuronal death or loss in certain brain regions has been well characterized in animal models of transient global cerebral ischemia. However, selective neuronal death in transient focal cerebral ischemia needs more investigation. Therefore, in this study, we studied selective neuronal death in the striatum (caudate putamen) of rats subjected to 15 or 30 min middle cerebral artery occlusion (MCAO). Neuronal death occurred in the dorsolateral field, not in the medial field in 30 min, not 15 min, MCAO-operated rats 5 days after MCAO using neuronal nuclear antigen immunohistochemistry and Fluoro-Jade B histofluorescence staining. In this group, immunoreactivity of glial fibrillary acidic protein in astrocytes was hardly shown in the dorsolateral field, although the immunoreactivity increased in the medial field. In addition, immunoreactivity of ionized calcium binding adapter molecule 1 in microglia was dramatically increased in the dorsolateral, not in the medial, field only in 30 min MCAO-operated rats. Briefly, these results show that at least 30 min of MCAO can evoke selective neuronal death, astrocytic dysfunction and microglial activation in the dorsolateral field of the rat striatum and suggest that a rat model of 30 min MCAO can be used to investigate mechanisms of neuronal death and gliosis following brief transient focal cerebral ischemic events for acute transient ischemic attack.

Brain ischemic preconditioning protects against moderate, not severe, transient global cerebral ischemic injury.

Ischemic preconditioning (IPC) in the brain increases ischemic tolerance to subsequent ischemic insults. In this study, we examined whether IPC protects neurons and attenuates microgliosis or not in the hippocampus following severe transient global cerebral ischemia (TCI) in gerbils. Gerbils were assigned to 8 groups; 5- and 15-min sham operated groups, 5-min and 15-min TCI operated groups, IPC plus 5- and 15-min sham operated groups, and IPC plus 5- and 15-min TCI operated groups. IPC was induced by subjecting animals to 2-min transient ischemia 1 day before 5-min TCI for a typical transient ischemia and 15-min TCI for severe transient ischemia. Neuronal damage was examined by cresyl violet staining and Fluoro-Jade B histofluorescence staining. In addition, microglial activation was examined using immunohistochemistry for Iba-1 (a marker for microglia). Delayed neuronal death and microgliosis was found in the CA1 alone in the 5-min TCI operated group at 5 days post-ischemia, and, in the 15-min TCI operated group, neuronal death and microgliosis was shown in all CA areas (CA1-3) and the dentate gyrus. IPC displayed neuroprotection and attenuated microglial activation in the 5-min TCI operated group. However, in the 15-min TCI operated group, IPC did not show neuroprotection and not attenuate microglial activation. Our present findings indicate that IPC hardly protect against severe transient cerebral ischemic injury.

Sac-1004, a vascular leakage blocker, reduces cerebral ischemia-reperfusion injury by suppressing blood-brain barrier disruption and inflammation.

BACKGROUND: Blood-brain barrier (BBB) breakdown and inflammation are critical events in ischemic stroke, contributing to aggravated brain damage. The BBB mainly consists of microvascular endothelial cells sealed by tight junctions to protect the brain from blood-borne substances. Thus, the maintenance of BBB integrity may be a potential target for neuroprotection. Sac-1004, a pseudo-sugar derivative of cholesterol, enhances the endothelial barrier by the stabilization of the cortical actin ring. RESULTS: Here, we report on the protective effects of Sac-1004 on cerebral ischemia-reperfusion (I/R) injury. Treatment with Sac-1004 significantly blocked the interleukin-1ß-induced monolayer hyperpermeability of human brain microvascular endothelial cells (HBMECs), loss of tight junctions, and formation of actin stress fiber. Sac-1004 suppressed the expression of adhesion molecules, adhesion of U937 cells, and activation of nuclear factor-κB in HBMECs. Using a rat model of transient focal cerebral ischemia, it was shown that Sac-1004 effectively ameliorated neurological deficits and ischemic damage. In addition, Sac-1004 decreased BBB leakage and rescued tight junction-related proteins. Moreover, the staining of CD11b and glial fibrillary acidic protein showed that Sac-1004 inhibited glial activation. CONCLUSIONS: Taken together, these results demonstrate that Sac-1004 has neuroprotective activities through maintaining BBB integrity, suggesting that it is a great therapeutic candidate for stroke.